This invention relates to electrophoresis generally, and more particularly, to an apparatus and method for performing capillary array electrophoresis on microfabricated structures.
In many diagnostic and gene identification procedures such as gene mapping, gene sequencing and disease diagnosis, deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or proteins are separated according to their physical and chemical properties. In addition to DNA, RNA or proteins, other small molecule analytes may also need to be separated.
One electrochemical separation process is known as electrophoresis. In this process, molecules are transported in a capillary or a channel which is connected to a buffer-filled reservoir. An electric field in the range of kilovolts is applied across both ends of the channel to cause the molecules to migrate. Samples are typically introduced at a high potential end and, under the influence of the electric field, move toward a low potential end of the channel. After migrating through the channel, the separated samples are detected by a suitable detector.
Typically, electrophoretic separation of nucleic acids and proteins is carried out in a gel separation medium. Although slab gels have played a major role in electrophoresis, difficulties exist in preparing uniform gels over a large area, in maintaining reproducibility of the different gels, in loading sample wells, in uniformly cooling the gels, in using large amounts of media, buffers, and samples, and in requiring long run times for extended reading of nucleotides. Moreover, slab gels are not readily amenable to a high degree of multiplexing and automation. Recently, micro-fabricated capillary electrophoresis (CE) devices have been used to separate fluorescent dyes and fluorescently labeled amino acids. Additionally, DNA restriction fragments, polymerase chain reaction (PCR) products, short oligonucleotides and even DNA sequencing fragments have been effectively separated with CE devices. Also, integrated micro-devices have been developed that can perform polymerase chain reaction amplification immediately followed by amplicon sizing, DNA restriction/digestion and subsequent size-based separation, and cells sorting and membrane lysis of selected cells. However, these micro-fabricated devices only perform analysis on one channel at a time. For applications such as population screening or DNA sequencing, such a single channel observation and analysis results in an unacceptable delay for screening many members of a population.
The invention provides a capillary array electrophoresis (CAE) micro-plate. The micro-plate has an array of separation channels connected to an array of sample reservoirs on the plate. The sample reservoirs are organized into one or more sample injectors. A waste reservoir is provided to collect wastes from sample reservoirs in each of the sample injectors. Additionally, a cathode reservoir is multiplexed with one or more separation channels. An anode reservoir which is common to some or all separation channels is also provided on the micro-plate. Moreover, the distance from the anode to each of the cathodes is kept constant by deploying folded channels. The corners on these turns may be right angle turns or more preferably, smooth curves to improve electrophoretic resolution..
In one aspect, the reservoir layout on the substrate separates the sample reservoirs by a predetermined spacing to facilitate the simultaneous loading of multiple samples.
In another aspect, cathode, anode and injection waste reservoirs are combined to reduce the number of holes N in the substrate to about 5/4N where N is the number of samples analyzed.
In another aspect, the separation channels are formed from linear segments.
In another aspect, the separation channels are formed from curvilinear segments, which may include radial segments.
In yet another aspect, the separation channels span from the perimeter of the plate to the central region of the plate. The separation channels may span the plate in a linear or a radial fashion.
In yet another aspect, a CAE micro-plate assembly is formed using a micro-plate, a reservoir array layer, and an electrode array. The assembly simplifies sample handling, electrode introduction and allows an increased volume of buffer to be present in the cathode and anode reservoirs.
Advantages of the invention include the following. The micro-plate of the present invention permits analysis of a large number of samples to be performed at once on a small device. Moreover, the micro-plate allows samples to be easily loaded while minimizing the risk of contamination. Additionally, the micro-plate is easy to electrically address. Further, the micro-plate supports a wide variety of formats that can provide higher resolution separation and detection of samples, faster separation and detection of samples, or separation and detection of more samples.
Other features and advantages will be apparent from the following description and the claims.